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Overview of Next-Generation Sequencing UNIT 7.31
Technologies
Barton E. Slatko,1 Andrew F. Gardner,1 and Frederick M. Ausubel2,3
1
New England Biolabs, Ipswich, Massachusetts
2
Department of Molecular Biology, Massachusetts General Hospital, Boston,
Massachusetts
3
Corresponding author: ausubel@molbio.mgh.harvard.edu

High throughput DNA sequencing methodology (next generation sequencing;

NGS) has rapidly evolved over the past 15 years and new methods are con-
tinually being commercialized. As the technology develops, so do increases in
the number of corresponding applications for basic and applied science. The
purpose of this review is to provide a compendium of NGS methodologies and
associated applications. Each brief discussion is followed by web links to the
manufacturer and/or web-based visualizations. Keyword searches, such as with
Google, may also provide helpful internet links and information.  C 2018 by

John Wiley & Sons, Inc.

Keywords: next-generation sequencing r NGS r Sanger sequencing

How to cite this article:

Slatko, B. E., Gardner, A. F., & Ausubel, F. M. (2018). Overview of
next-generation sequencing technologies. Current Protocols in Molecular
Biology, 122, e59. doi: 10.1002/cpmb.59

FOUNDING METHODOLOGY UNIT 7.4 Although relatively slow by current

The founding methods in DNA sequencing
were the Sanger dideoxy synthesis (Sanger &
Coulson, 1975; Sanger, Nicklen, & Coulson,
1977; UNIT 7.4) and Maxam-Gilbert chemical
cleavage (Maxam & Gilbert, 1980; UNIT 7.5)
methods. The Maxam-Gilbert method is based
on chemical modification of DNA and subse-
quent cleavage of the DNA backbone at sites
adjacent to the modified nucleotides. Sanger
sequencing uses specific chain-terminating
nucleotides (dideoxy nucleotides) that lack a
3 -OH group. Thus, no phosphodiester bond
can be formed by DNA polymerase, resulting
in termination of the growing DNA chain at
that position. The ddNTPs are radioactively
or fluorescently labeled for detection in “se-
quencing” gels or automated sequencing ma-
chines, respectively. Although the chemistry
of the original Maxam-Gilbert method has
been modified to help eliminate toxic reagents,
the Sanger sequencing by synthesis (SBS)
dideoxy method has become the sequencing
standard.
The Sanger sequencing method was devel-
oped in 1977 and is described in detail in
NGS standards, improvements in the Sanger
chain termination methodology, automation,
and commercialization have enabled it to re-
main the most appropriate sequencing method
for many current applications. Specifically, the
replacement of ultrathin “slab gels” with mul-
tichannel capillary electrophoresis, the devel-
opment of automated refillable reusable cap-
illaries, and “electrokinetic” sample loading,
have all contributed to the enhanced speed
and ease of the Sanger process. The most
significant innovations in Sanger sequencing
have been: (1) the development of fluores-
cent (terminator) dyes, (2) the use of thermal-
cycle sequencing to reduce the quantity of
required input DNA and thermostable poly-
merases to efficiently and accurately incorpo-
rate the terminator dyes into the growing DNA
strands, and (3) software developments to in-
terpret and analyze the sequences. The leader
in automated Sanger sequencing is Applied
Biosystems (AB; now part of ThermoFisher).
The current commercialized AB sequencers
all utilize fluorescent dyes and capillary
electrophoresis (CE). The machines vary in
DNA Sequencing

Current Protocols in Molecular Biology e59, April 2018 1 of 11

Published online April 2018 in Wiley Online Library (wileyonlinelibrary.com).
doi: 10.1002/cpmb.59
Copyright C 2018 John Wiley & Sons, Inc. Supplement 122

capacity, 4 capillaries (SeqStudio Genetic
Analyzer), 8 to 24 (3500 Series Genetic
Analyzer), and 48 to 96 (3700 Series Ge-
netic Analyzer), for DNA sequencing or
fragment analysis protocols. All of these
sequencers generate 600-1000 bases of accu-
rate sequence. Although a variety of Sanger-
sequencing-based sequencing machines have
been introduced over the years, including in-
struments from Licor, Amersham, MilliGen,
Perkin Elmer, and Dupont, all of them except
the AB machines have been discontinued.
The Sanger sequencing technology re-
mains very useful for applications where high
throughput is not required. Many DNA se-
quencing core facilities and sequencing-for-
profit companies provide Sanger sequencing
services. The most common uses are for in-
dividual sequencing reactions using a specific
DNA primer on a specific template, for ex-
ample to verify plasmid constructs or PCR
products. Now that molecular biology kits and
reagents for DNA purification and relatively
inexpensive high quality synthetic primers are
available from many vendors, even relatively
large Sanger sequencing projects can be com-
pleted in a reasonable time frame and cost.
In addition to sequencing DNA, another
useful application of capillary electrophore-
sis on the AB machines has been the
development of methods for measuring the
activity of selected enzymes acting upon flu-
orescently labeled DNA substrates, by anal-
ysis, for example, of DNA fragment size
(Greenough et al., 2016). Capillary elec-
trophoresis can also be used to simultane-
ously analyze multiple substrates, products
and/or reaction intermediates in a single reac-
tion using different fluorescent labels (Gree-
nough et al., 2016). For example, CE was
used in high-throughput studies of DNA poly-
merase and DNA ligase kinetics and coupled
enzyme pathways including Okazaki fragment
processing and ribonucleotide excision repair
(Greenough, Kelman, & Gardner, 2015; Scher-
merhorn & Gardner, 2015). AB CE is also
useful in glycobiology for analyzing fluo-
rescently labeled glycans (Callewaert, Gey-
sens, Molemans, & Contreras, 2001; Laroy,
Contreras, & Callewaert, 2006).
SECOND GENERATION
SEQUENCING METHODS
The term “next generation” has implied a
Overview of next step in the development of DNA sequenc-
Next-Generation ing technology and suggests there will be a
Sequencing
“next-next” generation naming of new tech-
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nologies in the future. We prefer to use the
term second generation, third generation, etc.,
realizing that the automated AB sequencing
machine technology described above is re-
ally the “second” generation after the orig-
inal Sanger methods using radioactivity and
slab gels. Given this naming convention, the
need for higher throughput sequencing of
large genomes at lower cost triggered the
development of many second-generation or
“nextgen” technologies using a variety of cre-
ative methodologies in addition to automated
Sanger sequencing. As with the commercial-
ization of automated Sanger sequencing, many
of these technologies are no longer in use (for
example, SolidTM , PolinatorTM HelicosTM ).
These “second” generation sequencing tech-
nologies and associated methods are described
below.
Second generation sequencing methods can
be grouped into two major categories, se-
quencing by hybridization, and sequencing by
synthesis (SBS). SBS methods are a further de-
velopment of Sanger sequencing, without the
dideoxy terminators, in combination with re-
peated cycles of synthesis, imaging, and meth-
ods to incorporate additional nucleotides in the
growing chain. At first glance, these new meth-
ods may seem expensive, but the reactions are
run in parallel often at nanoliter, picoliter, or
zeptoliter volumes in small chambers, and thus
the cost per base pair sequenced is nominal.
Continual refinements and miniaturization are
reducing costs even further.
A note about costs: costs for sequencing
encompass many variables, some of which
are often left out of commonly presented esti-
mates of “cost per base”. The reagent costs are
usually dependent on the volume ordered and
are often negotiated with the vendor. For ex-
ample, core facilities and sequencing centers
that order in larger quantities can obtain dis-
counted pricing. Costs usually do not include
labor and the bioinformatics pipeline at the
end of the process. Nevertheless, goals such as
the “$1,000 human genome” or reducing the
“cost per base” are gold standards to be met
by the sequencing technology and research
community.
Sequencing by Hybridization
This method was originally developed
in the 1980s, using arrayed DNA oligonu-
cleotides of known sequence on filters that
were hybridized to labeled fragments of the
DNA to be sequenced. By repeatedly hybridiz-
ing and washing away the unwanted non-
hybridized DNA, it was possible to determine
Current Protocols in Molecular Biology

whether the hybridizing labeled fragments
matched the sequence of the DNA probes on
the filter. It was thus possible to build larger
contiguous sequence information, based upon
overlapping information from the probe hy-
bridization spots. Sequencing by hybridization
has been largely relegated to technologies that
depend upon using specific probes to interro-
gate sequences, such as in diagnostic appli-
cations for identifying disease-related single-
nucleotide polymorphisms (SNPs) in specific
genes or identifying gross chromosome abnor-
malities (rearrangements, deletions, duplica-
tions, copy number variants or CNVs; Church,
2006; Drmanac et al., 2002; Hanna et al., 2000;
Mirzabekov, 1994; Qin, Schneider, & Brenner,
2012).
Sequencing by Synthesis (SBS)
SBS methods have taken several distinct
approaches (Fuller et al., 2009; Mardis, 2008;
Metzker, 2010; Quail et al., 2012; Shendure
& Ji, 2008). The “second generation” meth-
ods generally use a solid support containing
micro channels or wells in which the sequenc-
ing reactions occur. In general, most of these
new SBS methods do not use dideoxy termi-
nators, although “reversible” terminators are
used in some technologies, which allow the
nucleotide incorporation reactions to proceed
normally while imaging the incorporated nu-
cleotides, and then removing synthesis block-
ing moieties on the labeled nucleotides to al-
low the incorporation of the next base in the
sequence.
Current SBS methods differ from the ap-
proach of the original Sanger sequencing in
that they rely on much shorter reads (currently
up to about 300 to 500 bases). Further, they
generally have an intrinsically higher error
rate relative to Sanger sequencing, and rely
on high sequence coverage (“massively par-
allel sequencing”) of millions to billions of
short DNA sequence reads (50 to 300 nu-
cleotides) as a way to obtain an accurate se-
quence based upon the identification of a con-
sensus (agreement) sequence. However, for
some technologies, sequence context errors
occur and cannot always be corrected by in-
creasing the number of reads. For example, ho-
mopolymer sequences occur when DNA con-
tains consecutive multiples of the same base,
such as AAAAAAAAAA. In this case, se-
quencing platforms are limited in accurately
determining the number of consecutive bases.
Each platform generates its own unique set
of potential sequence context errors and users
need to be aware of these limitations. Using
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several platforms with different technologies
is often used to circumvent these issues.
The shift from “longer read lengths” to
“short read” technologies is now trending back
toward developing technologies that generate
longer primary read lengths, while maintain-
ing the “massively parallel” nature of the tech-
nology. This is occurring in the “third”, and
especially the “fourth” generation technolo-
gies, described below. This is fueled in part
by cost per reaction and in part by the desire
to obtain as much primary sequence read in-
formation as possible to circumvent sequence
context issues such repeated DNA elements.
Most SBS technologies utilize a method in
which the individual DNA molecules to be
sequenced are distributed to millions of sep-
arate wells or chambers, or tethered to spe-
cific locations on a solid substrate. The DNA
molecules, amplified by PCR or by isothermal
modified “rolling circle” amplification meth-
ods, are then subjected to DNA synthesis re-
actions in which labeled nucleotides, or chem-
ical reactions based on the incorporation of a
particular nucleotide, can be imaged or oth-
erwise detected. Many creative technologies
have been developed to enable the generation
of millions of DNA sequence reads in a single
sequence run. Sequence runs may last hours
or several days depending on the throughput.
454 Pyrosequencing
Although discontinued, we include this
as an example of the first of the “second”
generation sequencing methods that came to
market and utilized a novel approach, namely,
the detection of pyrophosphate, a byproduct
of nucleotide incorporation, to report whether
a particular base was incorporated in a grow-
ing DNA chain ((Ronaghi, Karamohamed,
Pettersson, Uhlen, & Nyren, 1996; see also
www.youtube.com/watch?v=WYBzbxIfuKs).
Individual DNA fragments, 400 to 700 base
pairs (bp) long are ligated to adapters and
amplified by PCR in an individual emulsion
“bead” (emPCR) reaction. DNA sequences
on the beads are complementary to sequences
on the adaptors, allowing the DNA fragments
to bind directly to the beads, ideally one
fragment to each bead.
DNA synthesis followed by chemical de-
tection of the DNA synthesis reactions then
occurs in a picoliter-sized chamber where py-
rophosphate release is measured. By consecu-
tively flooding the chambers with sequencing
reagents containing one of the 4 nucleotides,
when the correct nucleotide is incorporated
DNA Sequencing
in the synthesized strand, pyrophosphate
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release is measured utilizing a light-generating
reaction. The intensity of light also provides
information concerning homopolymer “runs”
of nucleotides in the sequence, although dif-
ficulties are encountered with larger tracts of
the same nucleotide. Pyrosequencing was de-
veloped in Sweden by Pyrosequencing AB,
and subsequently acquired by Qiagen who li-
censed it to 454 Life Sciences, before it was
ultimately acquired by Roche. 454 sequencers
were discontinued in 2013, although reagents
are still available from several suppliers. The
“454 sequencing” technology was commonly
used for genome sequencing and metagenome
samples because of the long read lengths (up to
600 to 800 nt) that are typically achieved and
relatively high throughput (25 million bases,
at 99% or better accuracy in a 4 hr run), facil-
itating genome assembly.
Ion Torrent (https://www.youtube.com/
watch?v=WYBzbxIfuKs)
Ion TorrentTM technology directly converts
nucleotide sequence into digital information
on a semiconductor chip (Rothberg et al.,
2011). In a DNA synthesis reaction, when a
correct nucleotide is incorporated across from
its complementary base in a growing DNA
chain, a hydrogen ion is released. This changes
the pH of the solution which can be recorded
as a voltage change by an ion sensor, much
like a pH meter. If no nucleotide is incorpo-
rated, no voltage spike occurs. By sequentially
flooding and washing out a “sequencing cham-
ber” with sequencing regents which include
only one of the 4 nucleotides at a time, volt-
age changes occur when the appropriate nu-
cleotide is incorporated. When two adjacent
nucleotides incorporate the same nucleotide,
two hydrogens are released and the voltage
doubles. Thus “runs” of a single nucleotide
can also be determined. Large homopolymer
strings of the same nucleotide are sometimes
difficult to discern, however.
Ion Torrent sequencing reactions occur in
millions of wells that cover a semiconductor
chip containing millions of pixels that convert
the chemical information into sequencing in-
formation. To begin the process, DNA is frag-
mented into 200 to 1500 base fragments which
are ligated to adapters. The DNA fragments
are attached to a bead by complementary se-
quences on the beads and adapters and are then
amplified on the bead by emPCR. This process
enables millions of beads to each have multi-
Overview of ple copies of one DNA sequence. The beads
Next-Generation are then flowed across the chip containing the
Sequencing
wells such that only one bead can enter an indi-
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vidual well. When the sequencing reagents are
then flowed across the wells, when the appro-
priate nucleotide is incorporated, a hydrogen
ion is given off and the signal recorded. A ma-
jor advantage of the system is that no camera,
light source or scanner is needed; nucleotide
incorporation is directly converted to voltage
which is recorded directly, greatly speeding up
the process.
The Ion Torrent system is sold by Thermo-
Fisher and several versions of the platform are
available, including the Ion Personal Genome
MachineTM (PGMTM ) System, Ion ProtonTM
System, Ion S5 system and ION S5 XL system,
each with different throughput characteristics.
An automated library and template prepa-
ration system is also available (Ion ChefTM ).
A large number of applications are supported,
including targeted and de novo DNA and RNA
sequencing, transcriptome sequencing, micro-
bial sequencing, copy number variation de-
tection, small RNA and miRNA sequencing,
and CHIP-seq (chromatin immunoprecipita-
tion sequencing; Furey, 2012).
Illumina Technology
By far the major player in the second gen-
eration sequencing arena is Illumina, using
technology first developed by Solexa and Lynx
Therapeutics. Illumina sequencing is based on
a technique known as “bridge amplification”
wherein DNA molecules (about 500 bp) with
appropriate adapters ligated on each end are
used as substrates for repeated amplification
synthesis reactions on a solid support (glass
slide) that contains oligonucleotide sequences
complementary to a ligated adapter. (See
www.youtube.com/watch?v=womKfikWlxM).
The oligonucleotides on the slide are spaced
such that the DNA, following being subjected
to repeated rounds of amplification, creates
clonal “clusters” consisting of about 1000
copies of each oligonucleotide fragment.
Each glass slide can support millions of
parallel cluster reactions. During the synthesis
reactions, proprietary modified nucleotides,
corresponding to each of the four bases, each
with a different fluorescent label, are incor-
porated and then detected. The nucleotides
also act as terminators of synthesis for each
reaction, which are unblocked after detection
for the next round of synthesis. The reactions
are repeated for 300 or more rounds. The use
of fluorescent detection increases the speed of
detection due to direct imaging, in contrast to
camera-based imaging.
Illumina sequencing supports a variety
of protocols including genomic sequencing,
Current Protocols in Molecular Biology

exome and targeted sequencing, metage-
nomics, RNA sequencing, CHIP-seq, and
methylome methods. Different Illumina
sequencing machines provide varying levels
of throughput, including the MiniSeq, MiSeq,
NextSeq, NovaSeq and HiSeq models. The
MiniSeq provides 7.5 Gb of information with
25 million reads/run in segments of 2 × 150bp
reads. The MiSeq can perform 2 × 300bp
reads, 25 million reads for an output of 15 Gb.
The NextSeq can provide 120Gb with 400
million reads at 2 × 150bp read length. Details
on each machine and its capabilities relative to
particular sequencing projects can be found in
https://www.illumina.com/systems/sequencing
-platforms.html.
One issue that can arise with Illumina se-
quencing is a lack of synchrony in the synthesis
reactions among the individual members of a
cluster, interfering with the generation of an
accurate consensus sequence and reducing the
number of cycles that can be performed. Care
also must be taken not to “overcluster” the
support, which requires accurate quantitation
of the amount of template DNA that is loaded
onto the array. Because of the large amount
of data generated, analysis of sequencing er-
rors generated from the sequencing process
can also be examined, aiding in the identifica-
tion of “real” sequence variants versus proto-
col induced artifacts; see for example (Chen,
Liu, Evans, & Ettwiller, 2016).
“THIRD” GENERATION (LARGE
FRAGMENT SINGLE MOLECULE)
SEQUENCING
In contrast to second generation sequenc-
ing methods, third generation sequencing
methods aim to sequence long DNA (and
RNA) molecules. The current commercialized
technology leader in this area is Pacific
Biosciences (PacBio) (https://www.youtube
.com/watch?v=v8p4ph2MAvI), which has
commercialized two sequencing systems,
the original RSII model and more recently,
the SequelTM (see https://www.pacb.com/pro
ducts-and-services/pacbio-systems/). PacBio
sequencing, also referred to as Single
Molecule Real Time (SMRT) sequencing,
enables very long fragments to be sequenced,
up to 30 to 50 kb, or longer. The SMRT
method involves binding an engineered
DNA polymerase, with bound DNA to be
sequenced, to the bottom of a well (zero-mode
waveguide, or ZMW, in a SMRT flow cell; see
https://www.pacb.com/smrt-science/smrt-sequ
encing/). A ZMW is a small chamber that
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guides light energy into an area whose
dimensions are small relative to the wave-
length of the illuminating light. Because of
the ZMW design and wavelength of light
utilized, imaging occurs only at the bottom
of the ZMW where the DNA polymerase,
bound to the DNA, incorporates each base
in a growing chain. The four nucleotides
are labeled with different phospho-linked
fluorophores for differential detection. When
a nucleotide is incorporated into the growing
chain, imaging occurs on the millisecond
time scale as the correct fluorescently-labeled
nucleotide is bound. After incorporation,
the phosphate-linked fluorescent moiety is
released, which “floats away” from the bottom
of the ZMW and can no longer be detected.
The next nucleotide can then be incorporated.
Imaging is timed with the rate of nucleotide
incorporation so that each base is identified
as it is incorporated into the growing DNA
chain. This simultaneously occurs in parallel
in up to one million zeptoliter ZMWs, present
on a single chip within the SMRT cells.
Template preparation is unique in the
PacBio process as it involves production of a
“SMRTbell”, a circular double-stranded DNA
molecule with a known adapter sequence com-
plementary to the primers used to initiate the
DNA synthesis on the template. This enables
the polymerase to read through large tem-
plates numerous times by traversing the cir-
cular molecule in each ZMW, until the poly-
merase stops, to build up a consensus sequence
(circular consensus sequence, CCS). As the
adapters ligated to each side of the insert each
have DNA synthesis priming sites, the se-
quencing polymerase can traverse the circular
SMRTbell in the 5 ->3 direction on either
DNA strand, providing complementary infor-
mation from both strands of the dsSMRTbell.
An important advantage of the PacBio
real time sequencing imaging and detection
process is that the rate of each nucleotide
addition during synthesis can be measured,
termed the inter-pulse duration (IPD). Many
(but not all) nucleotides with base modifi-
cations, such as some adenine and cytosine
methylations, change the IPD and thus can
be identified as a modified base (Fang et al.,
2012; Flusberg et al., 2010; Murray et al.,
2012; Rhoads & Au, 2015; Vilfan et al., 2013;
Zhang, Sun, Menghe, & Zhang, 2015; see
https://s3.amazonaws.com/files.pacb.com/png
/basemod_benefits_lg.png). Many different
modifications can be detected and catalogued
for epigenetic studies. At this point, not all
DNA Sequencing
modifications can be identified, including
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m5 C due to minor modification of the IPD.
However, chemical modification of such
nucleotides might enable their detection.
PacBio SMRT sequencing suffers from an
inherently high error rate, but this is usually
surmounted due to the depth of the number
of read passes obtained in each ZMW for
each SMRTbell template. Because errors are
stochastic rather than systematic, sequencing
the template multiple times and aligning the
individual sequence reads results in high ac-
curacy CCS reads. Further, similar sequenc-
ing of multiple templates provides additional
consensus.
PacBio SMRT sequencing offers several
advantages over previous methods. It enables
rapid identification of methylation sites for
epigenetic studies in addition to providing
long reads for genome assemblies. For exam-
ple, using PacBio technology, it is relatively
straightforward to assemble a complete bacte-
rial genome sequence using only a few SMRT
cells and determine the methylation patterns
within. Often, PacBio assemblies are com-
bined with other methods, such as Illumina
sequencing, for increased accuracy.
In terms of throughput, the PacBio RS II
(2013) uses chips with 150,000 ZMWs, which
when optimized, can generate as much as 350
megabases of sequence per SMRT cell. Op-
timization is carried out using a Poisson dis-
tribution so that only one polymerase bound
DNA molecule should be in each well. The
latest PacBio instrument, the Sequel, has 1 mil-
lion ZMWs and can generate 365,000 reads,
with average reads of 10 to 15 kb (7.6 Gb of
output). Continual upgrades of the chemistry
and technology (such as “magnetic bead load-
ing”, and use of the SAGE Pippin to isolate
large DNA fragments for PacBio sequencing
(see ancillary methods below) are designed to
provide more, longer, and more accurate reads.
TECHNOLOGIES ON THE
“FOURTH” GENERATION CUSP
It is possible to pass long DNA molecules
through small diameter “holes” and measure
differing currents as each nucleotide
passes by a linked detector (Benner
et al., 2007; Branton et al., 2008; Cherf
et al., 2012; Hornblower et al., 2007;
Kasianowicz, Brandin, Branton, & Deamer,
1996; Liu, Wang, Deng, & Chen, 2016; see
https://www.youtube.com/watch?v=GUb1TZv
Overview of MWsw). In theory, more than a hundred kb of
Next-Generation DNA could be threaded through the nanopore,
Sequencing
and with many channels, tens to hundreds of
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Gb of sequence could be achieved at relatively
low cost.
Two types of nanopore systems for DNA
sequencing are being developed, biological
membrane systems and solid-state sensor tech-
nology. Biological nanopore sequencing relies
on the use of transmembrane proteins em-
bedded in a lipid membrane to produce the
pores. Two proteins that have been utilized to
generate pores have been extensively studied:
alpha hemolysin and Mycobacterium smegma-
tis porin A (MspA). The rate of DNA passage
through the pores is regulated by the addition
of motor proteins, such as a highly processive
DNA polymerase (phi29) that ratchets DNA
through upon nucleotide addition. Other ac-
cessory proteins, such as a DNA helicase, ex-
onuclease I, or oligonucleotides to bind DNA
strands, enable unwinding and “ratcheting” of
the DNA nucleotides through the nanopore
for detection. DNA can be moved through the
pores at a constant rate for tens of thousands
of nucleotides. Solid state sensor technology
uses various metal or metal alloy substrates
with nanometer sized pores that allow DNA or
RNA to pass through.
Nanopore-based DNA sequencing was first
proposed in the late 1990s and commercial-
ization has recently been achieved by Oxford
Nanopore Technologies (ONT) with a portable
MinION (512 nanopore flowcell channels),
benchtop GridION (5 mimIONs in a single
module), and a high throughput PromethION
(in development, 48 flow cells of 3000
nanopores each; Greninger et al., 2015; see
https://nanoporetech.com/applications/dna-na
nopore-sequencing and https://nanoporetech
.com/how-it-works). These sequencers use
protein nanopores in an electrically resistant
polymer membrane through which character-
istic current changes occur as each nucleotide
passes thru the detector.
Long dsDNA molecules are first bound to a
processive enzyme, such as phi29 polymerase.
When the complex encounters a nanopore,
one DNA strand enters the nanopore and the
translocation rate through the pore is regulated
by DNA polymerase synthesis and translo-
cation. The processive enzyme enables the
DNA to be continuously and processively “ra-
cheted” through it. As a nucleotide passes
through the pore, it disrupts a current that
has been applied to the nanopore. Each nu-
cleotide provides a characteristic electronic
signal that is recorded as a current disrup-
tion event. Recording is in real time and while
10 kb reads are now a reasonable output, in
theory 100 kb of DNA can pass through each
Current Protocols in Molecular Biology

nanopore and be detected. Once DNA has left
a nanopore, the pore is available for use by a
different DNA molecule.
Because of its small, handheld size, the
MinION has potential for many applications
where portability and or space requirements
are at a premium. Currently the error rate is rel-
atively high but as with other high throughput
sequencing methods, this can be circumvented
by the large number of molecules that can
be sequenced. Nanopore technology has been
used to sequence environmental and metage-
nomic samples, is currently on the space sta-
tion, and has been used for bacterial strain
identification. Viral genomes, environmental
surveillance and haplotyping have been per-
formed using the MinION. Nanopore technol-
ogy is also able to identify base modifications,
similar to PacBio technology, enabling epige-
netic events to be readily identified. Nanopore
sequencing also offers direct RNA sequencing,
as well as PCR-free cDNA sequencing. Thus,
nanopore sequencing has the potential to offer
relatively low-cost DNA and RNA sequenc-
ing, environmental monitoring, and genotyp-
ing (Ammar, Paton, Torti, Shlien, & Bader,
2015; Cao et al., 2016; Loman, 2015; Schmidt
et al., 2017).
ANCILLARY METHODS FOR
HIGH THROUGHPUT
SEQUENCING AND COMPLETING
LARGE GENOME PROJECTS
Optical Mapping
Despite the recent advances in high
throughput single molecule long-read se-
quencing methods, additional methods may
be useful to complete or confirm the order
of various DNA contigs (contiguous pieces)
in a genome. The most popular of these meth-
ods is “optical mapping”, using labeling meth-
ods distributed along long DNA molecules
at nucleotide positions based upon sequence.
These provide large scaffolds for “pinning”
known sequences onto genome maps. By cre-
ating a “visual physical map” along large
DNA molecules (similar in principle to a
restriction enzyme map), one can correlate
DNA sequence with physical location. The
method was originally developed by David C.
Schwartz in the 1990s (Cai et al., 1995; Jing
et al., 1998; Meng, Benson, Chada, Huff, &
Schwartz, 1995; Schwartz et al., 1993), but
other methodologies have also been used to
create the optical maps. The use of nanofludic
methods, novel ways of visualizing and identi-
fying specific sites in the DNA molecules, and
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19343647, 2018, 1, Downloaded from https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/cpmb.59 by NRI FOR BIOMEDICAL SCIENCE AND, Wiley Online Library on [13/10/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
bioinformatics-based image capturing/ analy-
sis of fragments have led to improvements in
the technology.
Optical mapping is useful for completing
large genome projects (chromosome sized-
contigs) and other applications, such as the
identification of rearrangements in genomes. It
can be used to assist in genome assembly, com-
pare genomic structures, and correct genome
assembly errors. It can also be used for com-
paring strain differences, for instance in medi-
cal microbiology applications. A major advan-
tage of this method is that it avoids cloning or
PCR artifacts and analyzes a single molecule
at a time. Nevertheless, artifacts can be intro-
duced, and thus multiple fragments are utilized
to build a consensus map. The method has
advantages over the (previous) industry stan-
dard of pulse field gel electrophoresis (PFGE)
for creating large DNA fragment chromosome
maps. Optical mapping is less time consum-
ing and provides, in addition to fragment siz-
ing information, “roadmap” locations that can
be used for building consensus chromosome
maps (Bogas et al., 2017; Lam et al., 2012;
Mak et al., 2016; Mostovoy et al., 2016; Muller
& Westerlund, 2017; Neely, Deen, & Hofkens,
2011).
Generally, optical mapping begins by
shearing genomic DNA to a large size (100 mb
to 1 gb). The key is to label these large DNA
fragments at specific internal sites so that the
labeled molecules can be imaged and lined
up to provide contiguous overlapping maps.
A number of approaches can be used to label
specific genomic locations including digestion
of the DNA with limited single-stranded nick-
ing or a rare-cutting restriction enzyme or a
set of enzymes which cleave at specific diges-
tion sites. Labeling then can be performed on
the free ends with specific dyes or by enzy-
matic “fill–in” reactions using single-stranded
nucleases and polymerization dye incorpora-
tion. Enzymes which bind DNA at specific
sites can also be used (such as DNA methyl-
transferases) where a label can be transferred
to the DNA from the enzyme after binding.
Labeling A+T or G+C rich DNA sites can
also be used. The resulting fragments can be
attached to the surface of glass slides or in
elongated linear chambers for imaging using
appropriate microscopy.
Once positioned on the solid substrates,
the molecules are physically elongated. They
can then be sized and the positions of the
label(s), imaged, and recorded, creating an
“optical map” of that molecule. By combining
DNA Sequencing
images from numerous DNA molecules, an
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Supplement 122

overlapping large “consensus optical map”
can be made, which can then be used as a scaf-
fold for the pinning of other physical markers,
sequences, or contigs. A commercial leader in
this field is Bionano Genomics (San Diego)
(https://bionanogenomics.com/products/). Op
Gen (https://www.opgen.com/about-us/opgen-
overview/) will process samples as a commer-
cial vendor.
Electron Microscopy DNA Sequencing
Electron microscopy DNA sequencing is
another single-molecule sequencing technol-
ogy, first considered in the 1960s and 70s. To
be visualized, the DNA must be labeled with
heavy atoms as the electron microscope cannot
visualize individual nucleotides containing the
standard carbon, hydrogen, nitrogen, oxygen,
and phosphorus, isotopes in DNA. For these
methods to work, the DNA must be denatured,
labeled and stretched out on an electron mi-
croscope grid, using a “hypophase” method to
keep the DNA denatured and linear. In the-
ory, transmission electron microscopy DNA
sequencing could provide extremely long read
lengths, but the issue of electron beam damage
has not been solved. A major company in this
area is ZS Genetics (Wakefield MA) whose
technology involves DNA nucleotides labeled
with three heavy atom labels: bromine, iodine
or trichloromethane. These appear as differen-
tial dark and light spots on the micrograph and
the fourth DNA base is unlabeled. Sequenc-
ing by electron microscopy has not yet been
commercialized.
ANCILLARY TECHNOLOGIES
DNA Shearing
A key step for all DNA sequencing methods
is fragmenting DNA into a defined size. A
number of shearing and large fragment DNA
devices are available for isolation of DNA of
various sizes for DNA library construction.
Covaris, Inc. Shearing
(https://covarisinc.com)
Covaris, Inc. provides several acoustic
shearing devices and consumables to enable
fragmenting DNA and RNA to appropriate
sizes for DNA and fragment library prepa-
ration (150 bp to 5 kb) and also provides
protocols for chromatin and RNA shearing.
For larger fragment isolation, they provide a
G-TUBETM , designed to shear DNA into large
Overview of fragments with a mean size ranging from 6 kbp
Next-Generation
to 20 kbp at room temperature (20°- 30°C).
Sequencing
The g-TUBE works with a bench top cen-
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trifuge and the final size of the DNA frag-
ments is controlled by the acceleration rate and
speed of the centrifuge. g-TUBE uses centrifu-
gal force (the “g” in G-TUBE) to push the sam-
ple through a precisely manufactured orifice.
This produces shearing forces in the sample
that fragment the DNA.

R
Diagenode, Inc. Megaruptor
The Megaruptor was designed to provide
a simple, automated, and reproducible de-
vice for the mechanical fragmentation of DNA
from 2 kb to 75 kb. Shearing performance is
independent of the source, concentration, tem-
perature, or salt content of a DNA sample. The
software allows two samples to be processed
sequentially without additional user input and
without cross-contamination. Several compo-
nents for isolation of different sized DNA frag-
ments are available. The Megaruptor base unit
consists of an automated syringe pump with
attached 9-port ceramic distribution valve and
an integrated power supply. In order to control
the device, a laptop with pre-loaded software
is provided.
Enzymatic Fragmentation (NEBNext
Ultra II FS; New England Biolabs
Product Number E7805)
The NEBNext Ultra II FS enzyme in the
NEBNext Ultra II FS DNA Library Prep Kit
for Illumina contains the enzyme mix required
to shear a broad range of input amounts of
DNA into fragments for NextGen sequencing
on the Illumina platform. The protocols use a
minimum of 100 pg and create fragment sizes
100 bp to 1 kb. The reaction is generally 5 min,
with a 30 min heat enzymatic heat kill step at
65°C.
Sage Science Pippin (www.sagescience
.com/applications/dna-sequencing)
A novel technology for generating DNA
fragments for sequencing has been developed
by Sage Sciences. DNA from 100 bp to up
to 2 mb can be size selected and isolated
via a modified electrophoresis device that col-
lects the selected DNA size in mini-chambers.
The method can be used for any technique
requiring size selected DNA, including li-
brary construction, removing low molecular
weight content for long-read sequencing, and
preparing ultra HMW DNA libraries for long-
range genomic applications. Several versions
of their machines are available, the Pippin
Prep, BluePippin, or PippinHT, in which frag-
ments of selected lengths are collected, elimi-
nating all others.
Current Protocols in Molecular Biology

FUTURE OUTLOOK
As DNA sequencing technology advances,
the goal will be faster and more accurate se-
quencing (lower error rates, minimal artifacts)
with lower amounts of input DNA and RNA
at lower cost. Among these will be advances
in sequencing from single cells and from cir-
culating nucleic acids. Sequencing platforms
that are smaller, require less power (battery
operated), less reagents (zeptoliters or perhaps
even just a few molecules of input reagents)
and maintenance (perhaps disposable) will be
utilized in medical, agricultural, ecological,
and other settings (Shendure et al., 2017).
Higher order multiplexing (barcoding) will en-
able more samples to be processed in a shorter
time and at reduced cost. At the front end,
advances in robotics, liquid handling, sample
processing (nucleic acid preparation) will con-
tribute to these advancements. Equally impor-
tant will be advances in faster and more accu-
rate bioinformatic data analysis as well as data
transfer and storage.
ACKNOWLEDGEMENTS
F.M.A.’s laboratory is funded in part
by NIH grants P30 DK040561 and P01
AI083214.
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